• Up-regulation and co-localization of intestinal proteinase-activated receptor (PAR)-2 and Zot/zonulin receptor in active celiac disease

      Clemente, Maria Grazia; De Virgiliis, Stefano; Musu, Maria Paola; Usai, Paolo; Porqueddu, Patrizia; Cicotto, Lucia; Massidda, Carlo; Fasano, Alessio (2004)
    • Abstract: Celiac Disease Among Schoolchildren in Egypt: Results of a Pilot Study

      Catassi, C. (Carlo); Abu-Zakry, Mona; Kryszak, Deborah; Fasano, Alessio (2004-04)
    • Celiac Disease Case-Finding In Primary Care In North America

      North America Consortium Celiac Disease Case Finding; University of Maryland, Baltimore. School of Medicine. Center for Celiac Research (2004-04)
    • Proteinase-Activated Receptor 2 (PAR-2) Involvement In The Zot/Zonulin-Mediated Regulation Of Intestinal Tight Junctions

      Clemente, Maria Grazia; Vogel, Stefanie N.; Hollenberg, Morley D., 1942-; Fasano, Alessio (2004-05)
    • Zonulin Is Structurally And Functionally Related To The Mucosal Mast Cells-Derived Mast Cell Protease II

      Adams, David R., M.D., Ph.D.; Fang, Eugene; Vogel, Stefanie N.; Fasano, Alessio (2004-05)
    • Genetics and Epidemiology of Celiac Disease

      Fasano, Alessio (2004-05)
      Handout for the 2004 American Gastroenterological Association Digestive Disease Week. Author affiliation: Mucosal Biology Research Center and Center for Celiac Research, University of Maryland School of Medicine
    • Celiac Disease In Egypt: Preliminary Results Of A Pilot Screening Study In Schoolchildren

      Catassi, C. (Carlo); Abu-Zakry, Mona; Kryszak, Deborah; Fasano, Alessio (2004-05)
    • Intestinal Proteinase-Activated Receptor (PAR)-2 Is Up-Regulated In Active Celiac Disease and Co-Localize With Zot/Zonulin Receptor

      Clemente, Maria Grazia; De Virgiliis, Stefano; Musu, Maria Paola; Usai, Paolo; Porqueddu, Patrizia; Cicotto, Lucia; Massidda, Carlo; Fasano, Alessio (2004-05)
    • Effect Of Gluten on Intestinal Mucosal Biology: Studies on Cell Lines and Human Intestinal Biopsies

      Clemente, Maria Grazia; Drago, Sandro; El Asmar, Ramzi; Di Pierro, Mariarosaria; Sapone, Anna; Thakar, Manjusha; Iacono, Giuseppe; Carroccio, Antonio; D’Agate, Cinzia; Not, Tarcisio; et al. (2005)
    • Toxicity of gluten traces in patients on treatment for celiac disease. Results of a prospective, placebo-controlled, double-blind, randomized study

      Catassi, C. (Carlo); Fabiani, Elisabetta; Iacono, Giuseppe; D’Agate, Cinzia; Francavilla, Ruggiero; Volta, Umberto; Accomando, Salvatore; Picarelli, Antonio; De Vitis, Italo; Bardella, Maria Teresa; et al. (2005)
    • Toxicity of gluten traces: the Italian study on gluten microchallenge

      Catassi, C. (Carlo); Fabiani, Elisabetta; Mandolesi, Alessandra; Bearzi, Italo; Iacono, Giuseppe; D’Agate, Cinzia; Francavilla, Ruggiero; Corazza, Gino R.; Volta, Umberto; Accomando, Salvatore; et al. (2005)
    • Effect Of the Gluten Free Diet on Serum Zonulin Levels and Autoimmune Biomarkers in Both Treated and Untreated Celiac Disease Patients

      Kryszak, Deborah; Neri, Elena; Palese, Teresa; Sapone, Anna; Counts, Donald R.; Not, Tarcisio; Catassi, C. (Carlo); Fasano, Alessio (2005)
      Background: It is well known that Celiac Disease (CD) can be associated with other autoimmune diseases (AD). It is however still unclear whether the CD-associated risk of other AD is related to ongoing gluten ingestion or simply depends on common genetic background. Zonulin, which is responsible for the modulation of intestinal permeability, is up regulated in CD and other AD, such as Type 1 diabetes. Our hypothesis is that the loss of barrier function secondary to zonulin increase in CD can be involved in the high risk for AD co-morbidity. Aim: To establish the changes of serum zonulin levels and serum autoimmune antibodies in patients with newly diagnosed CD and after treatment with the gluten-free diet (GFD). Patients and Methods: They were 54 patients diagnosed with CD (20 M and 34 F; mean age: 39y; biopsy-proven: 42/54). Associated AD were found in 8 subjects (2 Type 1 diabetes, 1 Graves’s disease, 5 rheumatoid arthritis). Serum samples were collected at diagnoses and after a mean period of 17 months of GFD (range 10-49). All serum samples were measured for auto-antibodies related to CD (anti-transglutaminase - tTG, anti-endomysial - EMA), Type 1 diabetes (IA-2: tyrosine phosphates, IAA: anti-insulin antibodies, GAD: glutamic acid decarboxilase), thyroiditis (TPO: thyreoperoxidase antibodies, TG: thyreoglobulin antibodies), and zonulin levels. Results: at CD diagnosis increased serum zonulin were found in 76 % and auto-antibodies were detected in 39 % (TPO: 21.7%, TG 19.6%, GAD 6.5%, ICA 4.4% and IA-2 2.5 %). After GFD, EMA and zonulin remained altered in 13% of patients, and tTG in 35% of the subjects. Some auto-antibodies decreased (TPO: 10.9%, GAD 4.4%), while other remained unchanged (TG 23.9%, ICA 4.4%, and IA-2 2.2 %). Seven out of 53 patients did not start the GFD. These subjects had altered zonulin, EMA, and tTG and 14% of them were auto-antibodies positive. In these subjects, both zonulin levels and serum auto-antibodies did not change at the follow-up evaluation. Conclusions: Untreated CD typically show zonulin up-regulation and increased prevalence of serum auto-antibodies. After treatment with GFD, serum zonulin levels tend to normalize, a situation that is associated with a decreased prevalence of some auto-antibodies (especially TPO). These results indirectly suggest that recovery of the intestinal barrier function can decrease the risk of associated autoimmune phenomena. These results also suggest that if the GFD is implemented early (less 30 years of age) the auto-antibodies will seroconvert, suggesting a possible protective roll against autoimmunity co-morbidity, if CD is diagnosed early and started on a GFD.
    • The Vibrio Cholera-generated Zonulin occludens Toxin (Zot) N-terminal Cleavage Site Contains a Protease-activated Receptor Activating Peptide (PAR-AP) That Retains Biological Activity on Intestinal Tight Junctions

      Thakar, Manjusha; Vogel, Stefanie N.; Kao, Joseph P. Y.; Fasano, Alessio (2005)
      Background : We have previously demonstrated that Zonulin occludens toxin (Zot) elaborated by Vibrio Cholera anchors to the bacterial outer membrane through its single spanning domain and undergoes to a Vibrio-specific cleavage at amino acid residue 288. The resulting 12 Kda c-terminal fragment is then released in the intestinal micromilieu were exerts its permeating effect on intercellular tight junctions (tj). The N-terminus of th cleaved Zot fragment contains a conserved 6-mer protease activated receptor (PAR)-activating peptide (AP) motif. Aim: We have previously reported that Zot/Zonulin receptor is similar to PAR2, we elected to establish whether the six-mer motif (called AT1002) retains the Zot biological activity on tj. Methods: Rat small intestine was mounted in ussing chambers and AT1002-induced changes in transepithelial electrical resistance TEER) monitored Rat epithelial cells (IEC6) were used to study the intracellular signaling, including Ca2+ release,changes in tj protein-protein interactions, and phosphorylation of tj proteins. Results: Rat small intestine exposed to AT1002 showed a significant reduction in TEER as compared to the negative control starting at 30 minutes and reaching a plateau after 120 minutes. At a time point coincident with the effect con TEER (30 min) AT1002 induced an increment in ZO-1 phosphorylation that was associated to a decrease in ZO1-occludin interaction and an increase in ZO1-ZO2 interaction. Addition of AT1002 to IEC6 cells did not cause any increase in intracellular Ca2+, while the PAR-AP caused a dose-dependent increased in intracellular Ca2+ that reached a plateau at 50 fym. Conclusions: The 6-mer synthetic peptide At1002 retained the Zot biological activity an intercellular tj and caused a decrease in TEER. This effect was not associated to Ca2+ release, however was related to changes in protein-protein interaction of tj elements following Z)1 phosphorylation.
    • A New marker in the diagnosis of coeliac disease

      Clemente, Maria Grazia; Mucosal Biology Research Center, University of Maryland School of Medicine (2005)
    • Serum zonulin and intestinal permeability before and after a gluten-containing meal in both Type 1 diabetes and in their relatives

      Sapone, Anna; Fasano, Alessio (2005)
      Background: The trigger of the autoimmune destruction of pancreatic beta cells in Type 1 diabetes (T1D) is unclear. One theory is that antigens absorbed through the gut may be involved. We have recently described a protein, zonulin, that opens intestinal tight junctions and allows paracellular absorption of macromolecules. We have reported that zonulin is increased in the serum of a subset of patients with T1D and first degree relatives. Aim: The objective of this study was to determine if dysregulation of the zonulin pathway, both at baseline and following a gluten-containing meal, is linked to increased intestinal permeability (IP) and is involved in the pathogenesis of T1D. Methods: After obtaining informed consent, blood was obtained from children with T1D, parents, and siblings. Zonulin was measured in the serum of children with T1D (n=11) and their first degree relatives (n=19), by sandwich ELISA. IP was determined by HPLC measurement of both serum (at baseline and every hour up to 5 hours) and urine (5 hours collection) lactulose (LA) and mannitol (MA) after subjects ingested the sugars test solution. Results were expressed as LA/MA ratio. Results: There was a significant increase in zonulin (53%) and IP (47 %) in subjects compared to the negative cutoff. There was a significant relationship between elevation of zonulin and increased IP (p=0.026) and between LA/MA ratio in post-prandial serum samples and LA/MA ratio in urine (p=0.01). Although there was no change in mean zonulin levels with a meal, all subjects for whom IP data is available had elevation of zonulin at baseline and post-meal, and all had an increase in IP 1-hour post-meal. The 1 h post-prandial IP increase was statistically higher in T1D subjects (5.6 folds) as compared to their relatives (2.7 folds, p<0.01). There was no relationship between serum glucose and zonulin levels. Conclusions: Zonulin is significantly correlated with increased IP in children with T1D and their first degree relatives. These data suggest that increased serum zonulin is indicative of increased gut leakiness in children with T1D. Studies of the temporal relationship of an increase in serum zonulin and the development of diabetes autoantibodies are planned to suggest a causative relationship in a subset of genetically susceptible individuals.
    • Inhibition of the zonulin pathway blocks the progression from pre-clinical autoimmunity to Type 1 diabetes in BB/wor rats

      Fasano, Alessio (2005)
      Background: We have previously studied that zonulin, a protein involved in tight junctions modulation, is up regulated in BB/wor rats and is responsible for the increased intestinal permeability (IP) typical of this animal model of autoimmunity. We have also demonstrated that by blocking the zonulin pathway before the onset of autoimmunity, the incidence of Type 1 Diabetes (T1D) can be prevented in 75% of the animals. Aim : To determine whether the progression of T1D can be blocked, even though the autoimmune process is already established. Methods: 30 BB/ Wor rats were randomized after immune seroconversion (average age 55 days) in a treatment group (N=20) that received autoclaved water supplemented with 3mg /ml of zonulin receptor blocker AT1001 and HCO3- 1.5g/100ml to buffer gastric acidity, and a placebo group (N=10) that received only water with HCO3-. Serum zonulin and autoantibody levels were monitored at the beginning of the study and at its endpoint. Water intake was monitored daily, weight gain and serum glucose levels were checked weekly. Rats with fasting blood glucose  250 mg/dl were considered diabetic and were sacrificed within 24 hours of reaching the diabetic status. Results: Six out of 10 (60%) untreated rats developed T1D, while only 7/20 (35%) of the AT1001-treated animal progressed to T1D. The average age of onset of T1D was 85.410.4 in the placebo group and 86.010.3 in the treated group. AT1001 treatment did not change the serum zonulin levels between beginning (average age 55 days) and the endpoint of the experiment (average age 100 days) of the experiments. Conversely, the anti-glutamic acid decarboxylase (GAD) antibodies were significantly reduced in AT1001-treated rats that did not develop T1D (0.870.35) compared to the treated animals with the disease developed (1.870.59; p<0.05) Conclusions: The blockage of the zonulin pathway in BB/wor rats at their preclinical autoimmune stage significantly reduced the progression to T1D. This decreased incidence of T1D was associated to a significant reduction of the anti-GAD antibodies following AT1001 treatment.